Emerging precision neoadjuvant systemic therapy for patients with resectable non-small cell lung cancer: current status and perspectives

Over the past decade, targeted therapy for oncogene-driven NSCLC and immune checkpoint inhibitors for non-oncogene-driven NSCLC, respectively, have greatly improved the survival and quality of life for patients with unresectable NSCLC. Increasingly, these biomarker-guided systemic therapies given before or after surgery have been used in patients with early-stage NSCLC. In March 2022, the US FDA granted the approval of neoadjuvant nivolumab and chemotherapy for patients with stage IB-IIIA NSCLC. Several phase II/III trials are evaluating the clinical efficacy of various neoadjuvant immune checkpoint inhibitor combinations for non-oncogene-driven NSCLC and neoadjuvant molecular targeted therapies for oncogene-driven NSCLC, respectively. However, clinical application of precision neoadjuvant treatment requires a paradigm shift in the biomarker testing and multidisciplinary collaboration at the diagnosis of early-stage NSCLC. In this comprehensive review, we summarize the current diagnosis and treatment landscape, recent advances, new challenges in biomarker testing and endpoint selections, practical considerations for a timely multidisciplinary collaboration at diagnosis, and perspectives in emerging neoadjuvant precision systemic therapy for patients with resectable, early-stage NSCLC. These biomarker-guided neoadjuvant therapies hold the promise to improve surgical and pathological outcomes, reduce systemic recurrences, guide postoperative therapy, and improve cure rates in patients with resectable NSCLC.


Introduction
Lung cancer is the leading cause of cancer-related death in the United States and worldwide [1,2]. Globally, lung cancer currently contributes to an estimated 2.3 million new cases and 1.8 million per year. In the United States, lung cancer occurs in approximately 236,000 patients with 130,000 deaths in 2020. Lung cancer causes more deaths than breast, prostate, colorectal, and brain cancers combined [1]. The survival rates differ among lung cancer by histological type, stage at diagnosis, and treatment [3,4]. Non-small cell lung cancer (NSCLC) comprises 80-85% of lung cancer, followed by 10-15% of small cell lung cancer, < 5% of lung carcinoid or other types of lung cancer. NSCLC can be further divided into adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and other rare histological types. The American Joint Committee on Cancer (AJCC) is the most used staging system in clinical settings [5][6][7]. The 7th and 8th editions of AJCC are currently used in neoadjuvant and adjuvant clinical trials to describe stage distribution, prognosis, and treatment selection. Most patients with lung cancer are diagnosed at the incurable, advanced stage and contribute to the majority of lung cancer deaths. When left untreated, lung cancer progresses rapidly with a mean survival of about 7 months [8]. Improvement for patients with SCLC is limited, with a 5-year survival rate of 7% [9]. Over the past 3 decades, many advances have contributed to decreased lung cancer incidence and improved overall survival for patients with NSCLC [3,4]. These advances include primary prevention from smoking cessation, early detection of lung cancer in heavy smokers by lowdose chest CT scan, precise clinical staging through the use of positron emission tomography/computed tomography (PET/CT) scan, and pathological mediastinal staging, improved local therapy with surgery or radiation, the use of adjuvant chemotherapy, molecular targeted therapy and immunotherapy for patients with NSCLC, histology-directed systemic chemotherapy, tumor genotyping, increased use of molecularly targeted therapy and cancer immunotherapy [3,4]. Figure 1 summarizes the chronological milestones for the management of NSCLC. The 5-year relative survival of all lung cancers is 22% [1]. The 5-year survival for patients with stage I NSCLC is 68-92%, stage II 53-60%, stage IIIA 36%, IIIB-IV 0-26% [10]. The stage-based clinical management for NSCLC and the clinical efficacy of adjuvant systemic therapy with chemotherapy, targeted therapy, and ICI therapy, respectively, are summarized in Fig. 2. Recently, several clinical trials showed significantly improved clinical efficacy with promising increased cure rates using adjuvant and neoadjuvant targeted therapy and immune checkpoint inhibitors (ICI) after or with chemotherapy in patients with resectable NSCLC. This emerging precision neoadjuvant systemic therapy platform is an extension of precision medicine in lung cancer. However, clinical Fig. 1 The chronological milestones for the management of NSCLC. Major advances in systemic therapy with chemotherapy, targeted therapy and immunotherapy for NSCLC are illustrated. Beginning in 1990, chemotherapy was shown to be effective for prolonging survival in patients with NSCLC and platinum-based therapy became the cornerstone of treatment. Neoadjuvant or adjuvant chemotherapy modestly increased the overall survival by ~ 5% in patients with stage II-IIIA operable NSCLC. In 2004, targeted therapy erlotinib was approved for EGFR-sensitive NSCLC. During this time, researchers began utilizing modern gene sequencing programs to identify genetic markers for targeted therapeutics. The ALK gene rearrangement was identified and crizotinib was approved for ALK-positive tumors in 2011. In 2015, ICIs were found to improve survival and three immunotherapy agents were approved, including nivolumab, atezolizumab, and pembrolizumab. In 2018, a third-generation EGFR inhibitor osimertinib was approved as first line treatment for patients with metastatic NSCLC. By the end of 2020, adjuvant targeted therapy with osimertinib was approved for EGFR-sensitive NSCLC. In 2021, adjuvant immunotherapy with atezolizumab was approved for PD-L1 positive NSCLC. Neoadjuvant nivolumab and platinum-based chemotherapy becomes a standard of care for patients with stage IB-IIIA NSCLC without known EGFR or ALK genomic alterations in March 2022 implementation of this precision neoadjuvant treatment strategy requires multidisciplinary team coordination, extensive knowledge of tumor biology, and coordination of immune and molecular biomarker testing. In this article, we review these new developments, challenges in biomarker testing and endpoint selections, and practical considerations in neoadjuvant therapy for patients with operable, early-stage NSCLC.

Precision oncology in patients with non-operable mNSCLC
The landscape for the diagnosis and treatment of patients with non-operable, locoregionally advanced or metastatic NSCLC has evolved significantly in the past two decades [11] enabling the current era of precision lung cancer care. At diagnosis, NSCLC tumors are subjected to the multiplexed molecular and immune biomarker assays, as either companion or complementary diagnostics for targeted therapy or ICI treatment selection according to the National Comprehensive Cancer Network (NCCN) guidelines [12]. First, small molecular tyrosine kinase inhibitors (TKIs) effectively target a growing number of gain-of-function molecular targets (i.e., driver oncogenes), such as sensitizing mutations in the EGFR, BRAF V600E, MET exon 14 skip or ERBBR2 (HER2) genes, and gene rearrangements in the ALK, ROS1, RET, MET, and neurotrophic receptor tyrosine kinase (NTRK) genes, regardless of their tumor PD-L1 expression. These targeted therapies have improved the progression-free survival (PFS) and overall survival (OS) with favorable toxicity profiles compared to platinum-containing combination chemotherapy as first-line systemic therapy in 25-30% of non-operable NSCLC patients with variable oncogene driven mutations. Tumor genomic typing of NSCLC is important before starting the ICI therapy, not only because ICIs have no or inferior clinical effect in oncogene driven tumors, but because they have also been associated with increased incidence and severity of interstitial lung disease and other ICI-related toxicities when they are in sequential or concurrent use with EGFR TKIs in patients with metastatic NSCLC [13,14]. Secondly, in non-operable NSCLC patients with no EGFR or ALK genomic tumor aberrations, first-generation PD-1/PD-L1 inhibitors, either as monotherapy or in combination with platinum-containing combination chemotherapy, has become the standard of care first line treatment. Furthermore, adding anti-CTLA-4 to anti-PD-L1 alone or with chemotherapy confers additional clinical and longterm survival benefit in selected patient populations [15,16]. Uptake of ICI, which was only approved by the US Food and Drug Administration in 2015, has been rapid. In 2018, approximately 33% of patients with newly diagnosed, metastatic NSCLC received ICI, up from 12% in 2016 [10]. Despite these advances, the overall 5-year survival for SCLC is 7% and NSCLC is 26% overall and for all stages combined [10]. Nevertheless, the 5-year survival for nearly 50% of metastatic NSCLC patients with either oncogene-driven genomic alterations or immune checkpoint biomarkers have exceeded 15-20% in 2019 and > 25% in 2020 [17]. The expression of PD-L1 on the surface of tumor cells and immune cells, detected by immunohistochemistry, is the most used predictive biomarker used to guide treatment decisions with anti-PD-1 or anti-PD-L1 antibodies in patients with NSCLC. In addition, several tumor and blood molecular characteristics have been associated with increased clinical response to PD-1/PD-L1 inhibitors in NSCLC [18,19]. The clinical implementation of precision oncology in lung cancer requires close collaboration of multidisciplinary team, along with further technology and knowledge advances in understanding tumor biology and biomarker-guided treatment selection at the patient level.

Perioperative chemotherapy for resectable, early-stage NSCLC
Surgery remains the best treatment modality for curing patients diagnosed with resectable stage I-IIIA NSCLC [17,20]. The increased use of screening low-dose CT scans in high-risk patients for lung cancer has led to the identification of more early-stage patients who can be cured with local therapy [21]. In the United States, the proportion of lung cancer disease diagnosed at a localized stage increased from 17% during the mid-2000s to 20% in 2013 and 28% in 2018 [22]. However, surgery and general anesthesia can have severe complications, requiring assessments for pulmonary and cardiac function [5]. For those patients with inoperable early-stage NSCLC due to medical limitations in surgery, stereotactic body radiotherapy (SBRT) becomes a standard treatment with local control rates comparable to surgical series [23]. Up to 50% of NSCLC patients can have tumor recurrence after surgery [24], mostly from distant metastasis. Perioperative chemotherapy has been used to decrease tumor recurrence.
The goal of neoadjuvant or induction therapy is to reduce tumor burden prior to definitive therapy. Neoadjuvant cisplatin-based chemotherapy improves both survival and cure (5-year DFS) in unselected participants with stages IB-IIIA lung cancers to a degree at least equal to adjuvant use of the same chemotherapies [25,26]. A meta-analysis of 15 randomized controlled neoadjuvant trials of 2,385 patients demonstrated that OS was significantly improved with preoperative chemotherapy followed by surgery compared with surgery alone (HR, 0.87; 95% CI, 0.78-0.96; P = 0.007) [25]. Preoperative chemotherapy translated into a 5% absolute improvement in OS at 5 years. Recurrence-free survival (RFS) was also improved with neoadjuvant chemotherapy (HR, 0.85; 95% CI, 0.76-0.94; P = 0.002). The absolute increase in RFS at 5 years was 6%. Additionally, neoadjuvant chemotherapy decreased distant recurrence rate (HR, 0.69; 95% CI, 0.58-0.82; P < 0.0001). The absolute increase in freedom from distant recurrence at 5 years was 10%. Rates of complete pathologic response (pCR) are 5-10% and major pathologic response (MPR) are 11-20% with cisplatin-based chemotherapy [25,26]. The time to locoregional recurrence was not significantly improved with preoperative therapy, suggesting its OS benefit is mainly secondary to decreased rates of distant recurrence. As the neoadjuvant and adjuvant chemotherapy have similar modest 5-year survival benefit and low pCR (i.e., 5-10%) neoadjuvant chemotherapy has not been routinely used for the majority of early-stage NSCLC who are good surgical candidates. There is an unmet need to develop new systemic therapies to further improve the clinical outcomes of patients with resected NSCLC. Recently, perioperative targeted and immune checkpoint inhibitor therapies have been shown to significantly increase the pathological tumor response and recurrence-free survival without significant risk of tumor progression in patients with resectable NSCLC.

Recent advances of precision adjuvant systemic therapy
Recently, adjuvant targeted therapy with osimertinib for EGFR-sensitive NSCLC (ADAURA) [27] and immunotherapy with atezolizumab for PD-L1-positive NSCLC (IMpower010) [28] have also been shown to increase the survival, and become standard of care for patients with resected stage I-III NSCLC [29,30]. American Society of Clinical Oncology (ASCO) and NCCN have incorporated these data into their clinical practice guidelines for adjuvant systemic therapy for patients with resected stage I-III NSCLC [29,30]. For patients with Stage IB (3 < T ≤ 4 cm, N0M0) and sensitizing EGFR (Ex19del or L858R) mutations, 3-year adjuvant osimertinib is recommended without chemotherapy. For patients with stages IIA, IIB, and IIIA, adjuvant cisplatin-based chemotherapy is recommended for all fitted patients. Adjuvant osimertinib is recommended after chemotherapy for patients with tumors with sensitizing EGFR mutations, regardless of the PD-L1 status. Adjuvant atezolizumab is recommended for all patients with PD-L1 ≥ 1% after cisplatin-based chemotherapy except for patients with sensitizing EGFR mutations. The clinical uptake of these practice guidelines is limited.
However, there are still many unanswered questions for optimizing adjuvant systemic therapy for resectable NSCLC. First, several molecular risk stratification assays and novel cancer biomarkers, independent of clinical characteristics, are available to determine the risk of cancer recurrence and select high-risk patients for adjuvant chemotherapy [31][32][33]. Their roles in predicting clinical response to adjuvant immunotherapy needs to be studied. Second, given that neoadjuvant nivolumab and chemotherapy received FDA approval for in March 2022, it remains to be seen if adjuvant or neoadjuvant chemoimmunotherapy provides improved survival or even cure in patients with resectable NSCLC. Third, the role of PD-L1 IHC in selecting NSCLC patients for adjuvant ICI therapy remains unclear. In an interim analysis of a phase III trial (PEARLS/KEYNOTE-091 study), one-year pembrolizumab treatment significant improved median DFS over placebo (53.6 versus 42.0 months; HR, 0.76; 95% CI, 0.63-0.91; P = 0.0014) in patients with stage IB-IIIA NSCLC as an adjuvant treatment following surgical resection (lobectomy or pneumonectomy), regardless of PD-L1 expression [34,35]. In the TPS ≥ 50% subgroup, median DFS had yet to be reached in either treatment arm. At the time of this analysis, DFS events were observed in 32% of the pembrolizumab group and 38% of the placebo group. The KEYNOTE-091 study did not mandate adjuvant chemotherapy, and approximately 14% of patients did not receive adjuvant chemotherapy. Patients who received adjuvant chemotherapy and those with squamous histology had numerically better DFS with placebo. Thus, chemotherapy seems to be essential to the effectiveness of ICI. Ongoing phase III Chemo-IO (ACCIO) study aims to determine the role of adjuvant pembrolizumab with or after chemotherapy in resected NSCLC [36].

The immunosuppressive effect of surgery during peri-operative period
Surgery and anesthesia induce various metabolic and endocrine responses, which result in a generalized state of immunosuppression in the immediate post-operative period (days to weeks). Since initial reports of the prometastatic effects of surgery in early 1900s [37], numerous studies have been conducted to understand the mechanisms of surgery-induced immunosuppression in the development of cancer metastasis and poor clinical outcomes [38][39][40][41]. Surgery-induced prometastatic effect is linked to tumor cell dissemination to nearby blood vessels, organs, and lymphatics [42]. In addition, there are local and systematic release of growth factors (e.g. VEGF) [43], as well as impairment of innate and adaptive immunity [44][45][46]. Thus, the frequency of metastatic spread correlates with the degree of surgical trauma during the operation [39]. Adjuvant platinum-based systemic chemotherapy has been shown to decrease local and distant recurrence, presumably by eliminating factors that contribute to perioperative micrometastasis.
Activation of the sympathetic nervous system during surgery also has a profound deleterious effect on the immune system. Central neuroendocrine hormonal responses occur in response to trauma. Immune organs or lymphoid organs are innervated with nerve fibers that are directly stimulated by the sympathetic nervous system. When stimulated, there is a release of catecholamines which have an immunosuppressive effect [47]. In addition, stress activates the hypothalamic-pituitaryadrenal axis (HPA), releasing adrenocorticotropic hormone (ACTH) and glucocorticoids, mediating the effect of surgery on the immune system [48]. These effects may persist for several days following surgery and is proportional to the severity of the surgical stress. Additional factors, such as intraoperative blood loss, hypothermia and postoperative sepsis, also contribute to postoperative immune suppression [49]. Compared to conventional open surgery, minimally invasive techniques decrease the degree of surgical trauma and preserves lymphocyte subpopulations, neutrophil function, and cell mediated immunity. They also contribute to reduced post-operative complication rate and shorter hospital stays [38,50].

Preclinical rationales of neoadjuvant immunotherapy
As lung cancer and surgical intervention can be prometastatic and immunosuppressive, neoadjuvant immunotherapy can attack micromestasis early and mitigate its immunosuppressive effects to improve surgical and survival outcomes [41,51]. ICIs are designed to activate exhausted tumor-reactive T cells, which are responsible for killing tumor cells. Current data suggest that the presence of high membranous PD-L1 IHC staining on tumor cells and the presence of intratumoral PD-1 expressing tumor infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are favourable prognostic factors and the best predictive factors of clinical response to ICIs [19]. A T-cell inflamed gene expression profile (GEP) in addition to PD-L1 IHC has improved the prediction of favourable clinical response to ICIs [19]. ICIs could increase the number of absolute lymphocyte counts (ALCs), restore the function in exhausted CD8 + T cells and induce phenotypical and functional changes of effector immune cells [52]. Recent studies showed that early stage and late-stage NSCLC have distinct tumor biology [53]. Neoadjuvant or preoperative injection of anti-PD-1 inhibitors might be advantageous to activate tumor infiltrating T-cells prior to surgery and to avoid the reduction of PD-1 expression on immune cells in the postoperative period. Table 1 summarized the reported neoadjuvant trials in patients with early-stage NSCLC. Over 80% of NSCLC patients were able to have complete tumor resection. Unlike the trials conducted in patients with non-operable, locally advanced NSCLC, ICI monotherapy seems to have less effect compared to the ICI and chemotherapy combination as evaluated by pCR and MPR (Fig. 3). Taking nivolumab as an example, the clinical efficacy is increased when chemotherapy was added. In the first pilot study [54], neoadjuvant nivolumab monotherapy (given at a dose of 3 mg/kilogram of body weight every 2 weeks for 2 doses) was well tolerated without any new adverse events (perioperative adverse events of grade 3 or 4 for 90 days after the administration of the last nivolumab dose) and did not delay surgery (as defined by no more than 37 days). Of the 21 patients who had evaluable radiographic results, 2 patients (10%) had a partial response, 18 (86%) had stable disease, and 1 (5%) had disease progression. Of the 21 tumors that were removed, 20 (95%) were completely resected and pathological down-staging from the pretreatment clinical stage occurred in 8 patients (40%). MPR was observed in 9 (45%) tumors. The tumor mutational burden but not PD-L1 expression by IHC was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in 8 of 9 patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a pCR on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab [54]. At a median of 12 months of postoperative follow-up (range, 0.8 to 19.7), 16 of 20 patients (80%) who had undergone surgical resection were alive and recurrence-free. The recurrence-free survival rate at 18 months was 73% (95% CI, 53 to 100).

Emerging data of neoadjuvant ICI and chemotherapy for non-oncogene-driven NSCLC
NEOSTAR was the first randomized phase II trial comparing nivolumab monotherapy and nivolumab and ipilimumab combination for 3 cycles before surgery [71]. Forty-four patients were enrolled in each arm. Compared with nivolumab monotherapy, nivolumab plus ipilimumab resulted in higher pCR rates (10% versus 38%), less viable tumor (median 50% versus 9%), higher MPR (50% versus 24%), and greater frequencies of effector, tissue-resident memory T cells [66]. High percentages of baseline tumor cells with expression of PD-L1 was positively correlated with radiographic responses and pathologic tumor responses at the time of surgery.
CheckMate 816 trial is the randomized phase III study evaluating neoadjuvant chemotherapy with nivolumab plus platinum-doublet chemotherapy in 358 newly diagnosed patients with resectable stage IB to IIIA NSCLC [73]. Patients with known sensitizing EGFR or ALK mutations were excluded. Patients were stratified by cancer stage, PD-L1 status, and gender. Patients were randomly assigned 2:1 to receive nivolumab at 360 mg every 3 weeks plus chemotherapy every 3 weeks (three cycles) versus the same chemotherapy schedule. Then, patients underwent radiologic staging and surgery within 6 weeks of neoadjuvant therapy. They had the option of adjuvant therapy with or without radiation therapy. An exploratory arm of nivolumab plus ipilimumab was closed early. The primary endpoint was pCR by blinded independent  Table 2 and  Table 3 summarize ongoing neoadjuvant clinical trials containing either ICI alone or with chemotherapy for NSCLC patients registered at clinicaltrials.gov., respectively. In addition of chemotherapy, many studies are evaluating ICI with either other immunotherapy, chemotherapy, or targeted therapy for patients with early-stage NSCLC as summarized in Table 4. Currently, there are no sufficient data to support the use of radiation as neoadjuvant therapy for resectable NSCLC. In a phase III trial of induction chemoradiation in patients with resectable, stage IIIA/N2 NSCLC, radiation did not add any benefit to indication chemotherapy followed by surgery [74]. In a small, randomized, phase II study (N = 30 each arm), the addition of 2 cycles of neoadjuvant durvalumab to stereotactic body radiotherapy (SBRT) to primary tumor was well tolerated and associated with a high MPR rate compared to SBRT alone (53.3% vs 6.7%; HR, 16.0; 95% CI, 3•2-79•6; p < 0.0001) in patients with resectable stage I-IIIA NSCLC [75]. Ongoing studies are evaluating the role of radiation in neoadjuvant ICI therapy in resectable NSCLC (Table 5).

Neoadjuvant molecularly targeted therapies for oncogene-driven NSCLC
New generation targeted therapies have improved clinical outcomes in non-operatable metastatic NSCLC patients who were adherent to NCCN-recommended biomarker testing and first-line targeted therapy [77]. Asian patients with adenocarcinoma have higher prevalence of unique biologic features such as a higher incidence of oncogene-driven NSCLC (mainly EGFR-mutant and ALKrearranged tumors), and the majority of Asian women with lung cancer are never-smokers. For patients with metastatic NSCLC, tumor genomic typing of NSCLC is important before starting the ICI therapy, not only because ICIs have low or inferior effect in EGFR-mutant or ALK-rearranged NSCLC, but also they have been associated with increased incidence and severity of interstitial lung disease and immune-mediated adverse effects (including pneumonitis, colitis and hepatitis) when they are in sequential or concurrent use with EGFR TKIs in patients with metastatic NSCLC [13,78]. Given this concern, it is reasonable to perform genotyping of earlystage NSCLC tumors for targeted neoadjuvant therapy. Retrospective review of 40 patients with oncogene-driven NSCLC showed induction ICI therapy achieved a pCR rate of 12.5%, a MPR of 37.5% with a median EFS of 28.5 months [79]. NSCLC patients with sensitizing EGFR or ALK mutations were excluded in the CheckMate 816 trial [73]. Figure 4 summarizes the schema for an ongoing biomarker-driven precision neoadjuvant screening and matched targeted treatment trials for stage IA2-III NSCLC. The LCMC4 Evaluation of Actionable Drivers in EaRly Stage Lung Cancer (LEADER) screening trial (ClinicalTrials.gov Identifier: NCT04712877) builds on the Lung Cancer Mutation Consortium (LCMC)'s previous endeavors to now bring personalized neoadjuvant therapy to patients with early-stage lung cancers      Patients whose tumors harbor one of these oncogenic drivers are matched to one of the effective targeted therapies for patients with stage IV lung cancers. After 8 weeks of targeted therapy, patients will undergo surgical resection. Table 6 summarizes ongoing neoadjuvant clinical trials containing molecularly targeted therapeutics for patients with resectable NSCLC.

Pre-operative evaluation
Although lung resection is the cornerstone of curative treatment for lung cancer, there is an associated risk of mortality and morbidity with treatment. This is especially true for patients with impaired lung function, such as chronic obstructive pulmonary disease (COPD). Depending on the extent of lung resection, patients may have deterioration in their pulmonary function and may live with permanent changes that negatively impact their quality of life [80]. Therefore, comprehensive preoperative evaluation is an essential component to predict perioperative complications and long-term survival. Thoughtful consideration of surgical risks and benefits should be evaluated for each patient. The following section provides an overview of literature and a framework of current guidelines in the pre-operative evaluation process. Figure 5 summarizes the multidisciplinary evaluation for NSCLC patients for biomarker driven neoadjuvant therapy.

Radiographic evaluation for staging
Preoperative assessment begins with diagnostic imaging to localize the disease, visualize malignant involvement to adjacent structures, and determine the presence of metastasis. If an indeterminate nodule is seen on radiograph or chest CT, the first step is to compare the nodule with prior imaging. The most common staging system is the TNM staging system [7]. T refers to the size and extent of the main tumor (the primary tumor). N refers to the number of nearby lymph nodes that have cancer. M refers to whether the cancer has metastasized. Current recommendations state that further diagnostic testing is not warranted for solid nodules that have been stable for two years. On the other hand, if further work-up is indicated, combined 18 F-FDG PET and CT has emerged as a valuable tool that is integral for all patients undergoing surgical evaluation [81,82]. While CT scans can confirm the presence of disease, 18 F-FDG PET used in conjunction provides insight into mediastinal lymph nodes, distant metastasis, and metabolic activity of the cancer. PET scans have been associated with higher accuracy, with both higher sensitivity and specificity than CT scanning alone [82]. Combined 18 F-FDG PET/CT has been able to reduce the total number of thoracotomies and futile thoracotomies in NSCLC [83].

Tissue Biopsies
Histopathological evaluation of tumor biopsies is required not only for histologic diagnosis of lung cancer but also for molecular and immune biomarker analysis [84]. In the current era of precision lung cancer care, first-line systemic therapies for patients with metastatic NSCLC with good performance status are selected based on histology, and both molecular and immune biomarkers [85]. Furthermore, histopathological analysis of lymph nodes is essential for accurate pathological staging before surgery. Biopsy can be performed by bronchoscopy, mediastinoscopy, or needle biopsy [20]. Traditionally, mediastinoscopy has been the gold standard for pathological staging of mediastinal lymph nodes [86]. However, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as a well validated diagnostic tool demonstrated to have high accuracy. In patients with N2 disease, EBUS-TBNA had the ability for accurate nodal staging that could not be detected by either CT or PET/CT [87]. For patients undergoing diagnostic evaluation, EBUS-TBNA, compared to traditional bronchoscopy and transthoracic needle biopsy (TTNB), has been associated with lower complication rates, fewer required biopsies, and decreased need for repeat biopsies [88].

Cardiopulmonary Testing
Patients undergoing evaluation for resection often have underlying comorbidities. Cardiovascular diseases (CVDs) are the most frequently observed; this impacts up to 23% of patients with NSCLC and limiting survival [89,90]. Currently, the American College of Chest Physicians Evidence-Based Clinical Practice Guidelines recommend cardiovascular evaluation for all patients undergoing preoperative evaluation for risk stratification, determination of operability, and prediction of perioperative complications [91]. Cardiac assessment begins with a thorough evaluation of the patient's cardiac history and identification of risk factors that can predispose patients to adverse cardiac events. The thoracic revised cardiac risk index (ThRCRI) is a tool developed to evaluate cardiac risk specifically in thoracic patients undergoing lung resection and has shown to have good discrimination [92]. The ThRCRI is a weighted score of four risk factors: renal status (creatinine > 2 mg/dL), ischemic heart disease, cerebrovascular disease, and pneumonectomy [93]. Based on the cumulative score, patients can either proceed with surgical resection or undergo further evaluation to assess cardiac function. Any patient who has a ThRCRI score of ≥ 2, cardiac conditions that require monitoring, a newly suspected cardiac condition, and/or or an inability to climb 2 flight of stairs require evaluation by a cardiologist and optimization of cardiac function prior to surgery. In addition to a comprehensive cardiac evaluation, pulmonary function tests are a non-invasive approach to inform the extent of resection that is feasible and evaluate long-term survival [94]. The two primary metrics used to determine pulmonary function include FEV 1 and D LCO , as well as their respective predicted postoperative (PPO) values. PPO is calculated by estimating the proportion of the lung being resected and subtracting the value from baseline FEV 1 and D LCO . In patients with either PPO FEV 1 or PPO D LCO > 60%, they are considered low risk and can proceed to surgery without requiring further testing. On the other hand, if either the PPO FEV 1 or PPO D LCO < 60%, further evaluation of baseline cardiopulmonary function is recommended. Ensuring adequate cardiopulmonary reserves is critical as low FEV 1 , PPO FEV 1 , D LCO and PPO D LCO values have been associated with increased risk of morbidity and mortality [95,96]. Specifically, PPO FEV 1 and D LCO < 40% has been used as a cut-off value associated with an independent predictor of mortality with surgery [97].
In patients with diminished pulmonary function tests, the current recommendation is to pursue exercise testing and evaluate maximal oxygen consumption (VO 2max ). Determining baseline cardiopulmonary reserve is necessary, as patients with lung cancer often have low baseline exercise capacity due to underlying atherosclerotic disease and exposure to cigarette smoking. Decreased cardiopulmonary function at baseline is concerning, as lung function often decreases after resection [98]. This is compounded by the fact that patients are exposed to cardiotoxic treatments, causing radiation-induced cardiovascular disease and cardiac dysfunction associated with commonly used platinumbased antineoplastic agents [99]. While exercise capacity can be described subjectively, Cardiopulmonary Exercise Testing (CPET) is valuable as an objective measurement that can quantify cardiopulmonary function, subsequently allowing stratification of patients based on distinct cut-off values. Furthermore, CPET elucidates the effectiveness of the patient's compensatory mechanisms to maintain adequate circulation and oxygenation with increasing physiological stress. If CPET is unavailable, alternative validated methods that have been investigated include the 6-min walk test (6MWT), shuttle walk test (SWT), and stair-climbing test (SCT) [100].
In CPET, patients exercise with either a treadmill or cycle ergometry at incremental workloads while maximal oxygen consumption (VO 2max ), maximal carbon dioxide consumption (V CO2 ), minute ventilation (V e ), oxygen saturation, heart rate, and blood pressure are measured or calculated [101]. Among the variables measured, VO 2max has shown to be the greatest predictor of postoperative complications and used to determine surgical risk. In patients unable to achieve VO 2max , peak oxygen consumption (VO 2 peak ) can be used as a surrogate. A patient with VO 2max > 20 mL/kg/min is deemed low risk, 10-20 mL/kg/min is moderate risk, and < 10 mL/kg/min is high risk [100,102]. In patients with VO 2max < 10 mL/ kg/min, alternative therapies to surgery and/or minimal resection should be considered due to the high risk of mortality associated with major resection [103].
Other factors affecting surgery Lung cancer survival rates vary by geographic location, histologic subtype, stage at diagnosis, and treatment [2]. Many additional factors, such as the COVID pandemic, socioeconomic status, timely access to health care, area of residence, and immigration status can also contribute to differential incidence and mortality by race [104]. Stage III NSCLC is a heterogenous group of disease that has significant variations in the selection of treatment modality. Currently, only 21% of patients with stage III NSCLC undergo surgery, whereas most (61%) are treated with chemotherapy and/ or radiation [10]. In a recent report, surgery is substantially lower among Black patients with NSCLC with 49% for stages I-II and 16% for stage III compared to 55% and 22% for White patients, respectively [10]. This treatment disparity is also exacerbated by the fact that Black patients continue to be less likely to be diagnosed with stage I disease than White patients for most cancers. As the benefit of neoadjuvant systemic therapy is most prominent in patients with stage III NSCLC, this practice disparity needs to be considered.

Evaluation of neoadjuvant therapy
Although overall survival remains the gold standard of endpoints for neoadjuvant clinical trials, it will take a decade to mature. Several surrogate endpoints that have been used for evaluating neoadjuvant chemotherapy are being explored in the neoadjuvant ICI trials.

Surgical endpoints
The surgical endpoints for neoadjuvant therapy are feasibility of surgery, complete surgery, and minimally invasive surgery. Currently, more than one-half (55%) of patients with stage I or II NSCLC undergo surgery with either sublobar resection (partial removal of a lobe of the lung), sleeve resection (removal of the tumor and a portion of the affected airways), lobectomy (entire removal of an affected lobe), or pneumonectomy (removal of one lung). There are many factors to consider when discussing or planning for neoadjuvant therapy in lung cancer patients that should be evaluated. First is feasibility of surgery. We must consider patient factors which include, but are not limited to, comorbid conditions, pulmonary function, suitability for surgery, and overall functional status. Second, are tumor characteristics including location of the tumor. Is the tumor peripheral or central; will it require segmentectomy, lobectomy, bilobectomy, or pneumonectomy? In some cases, en bloc chest wall resection and reconstruction may need to be considered in order to achieve an R0 resection. In addition to these endpoints, there are other factors to consider, such as invasiveness of surgery. It is well known that there are several benefits and advantages that are attributed to minimally invasive approaches over thoracotomy which include video-assisted thoracic surgery (VATS) and robot-assisted thoracic surgery (RATS). These benefits include faster recovery time, shorter hospital stay, decreased pain, smaller incisions, and fewer complications.
Neoadjuvant therapy may lead to certain surgical challenges which have been reported such as the presence of significant inflammation and fibrosis which have ultimately led to longer operative times and more conversions from a minimally invasive to an open operation [105]. It is notable however, that this observation did not translate to a substantially higher rate of postoperative complications than for other studies with neoadjuvant chemotherapy [106]. In addition, immune-mediated side-effects, such as pneumonitis, can delay or prevent surgery, and can also complicate the postoperative period [66].

Clinical endpoints
The gold standard of neoadjuvant therapy is OS. EFS or DFS is a surrogate endpoint of OS for neoadjuvant chemotherapy, which takes a long time to mature. Several clinical trials demonstrated that pCR to neoadjuvant chemotherapy has been associated with improved survival in retrospective studies [107,108]. Based on these data, the US FDA has accepted pCR as an endpoint for the approval of new drugs in the neoadjuvant study. This strategy can markedly shorten the time and costs needed in the new drug development in the neoadjuvant setting. The role of EFS or DFS as the clinical endpoints for cure and OS remains to be defined in precision neoadjuvant targeted and immunotherapy for resected, early-stage NSCLC.

Pathological endpoint
Major pathologic response (MPR) was defined as a residual viable tumor of less than or equal to 10% [107]. Both pCR and MPR were established from the neoadjuvant chemotherapy trials and serve as surrogate endpoints for OS for patients with resectable tumors after neoadjuvant chemotherapy. Their roles in neoadjuvant immunotherapy need to be further defined [109]. 18 F-FDG PET/CT scan has been used in initial staging at diagnosis of early-stage NSCLC [81,82]. In radiographic evaluation for patients with advanced or metastatic NSCLC, ORR is the sum of CR and PR per Response Evaluation Criteria in Solid Tumors (RECIST) [110,111]. However, in neoadjuvant NSCLC studies, radiographic response by conventional CT or PET/CT scans seems to underestimate the tumor response by pathological evaluation (Fig. 6). This was first noticed in neoadjuvant nivolumab study, -3 patients achieved pCR, but none were evident by CT imaging; one patient even had apparent tumor growth radiologically. This phenomenon was called "pseudo progression" due to immune cell infiltrates in patients with advanced solid tumors receiving ICI therapy. In the phase III study of CM-816, ORR was higher than the sum of pCR and MPR [107,108]. Improved radiographic evaluation for predicting pathological response to neoadjuvant treatment is needed. Furthermore, the dynamic changes in cancer metabolism to targeted therapy, immunotherapy, or chemoimmunotherapy might be different. Ongoing studies are Fig. 6 Pathological and radiographic outcomes of nivolumab alone or in combination with ipilumumab or chemotherapy in early-stage and metastatic NSCLC. The pathologic complete response (pCR, solid bar in orange) and major pathologic response (MPR, solid bar in blue) rate (%) by histopathological evaluation were shown in resectable, early-stage NSCLC. No pathological stage is available for patients with unresectable NSCLC. The overall response rate (ORR) is the radiographic evaluation of tumor response by RECIST, which is higher in early-stage NSCLC compared to mNSCLC. Per RECIST V1.1, CR (dashed bar in orange) is defined as disappearance of all targeted lesions, and any pathological lymph nodes (whether target or non-target) must have reduction in short axis to < 10 mm. Partial response (PR, dashed bar in blue) is defined as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. This PR criteria correlate with at least a 50% decrease in tumor volume evaluating better tracers [109] for differentiating tumor cells from immune cells or sensitive scanner [112] to improve the in vivo assessment of cancer metabolism to precision neoadjuvant therapy in NSCLC.

Correlative studies Tissue biomarker endpoints
Although genotyping of plasma circulating tumor DNA (ctDNA) has received US FDA approval and has been increasingly used to complement tissue-based genomic assays in precision oncology, pathohistological assessment of tumor specimens remains the gold standard for diagnosis of lung cancer. High TMB and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse [113]. A meta-analysis included 10 studies involving 461 NSCLC patients. Compared with PD-L1 expression < 1%, PD-L1 expression ≥ 1% was associated with a higher rate of MPR and pCR. High TMB was also associated with MPR and pCR. Similar findings were observed in subgroup analyses, regardless of PD-1/PD-L1 inhibitor monotherapy or their combination with chemotherapy. Notably, 50% as the cutoff value for PD-L1 expression demonstrated better prediction efficacy for MPR than that of 1% [114]. A recent whole exosome analysis of 14 NSCLC patients (3 adenocarcinoma and 11 squamous cell carcinoma) who achieved MPR with chemoimmunotherapy, the detection of homologous recombination deficiency (HRD) was associated with enhanced response to neoadjuvant immunotherapy [115]. Further studies are needed to validate the results in prospective neoadjuvant immunotherapy studies.

Blood biomarker endpoints
Effective neoadjuvant therapy can result in insufficient viable tumor cells left in surgical specimens for biomarker testing and selection for adjuvant therapy. Increasingly, liquid biopsy and plasma ctDNA assays have been used as a minimally invasive way to study tumor biology and monitor dynamic changes of molecular and immune biomarkers during cancer treatment [116]. First, undetectable ctDNA has been associated with elimination of micrometastasis and clinical remission. In the CK-816 study, ctDNA clearance was higher in the 24 patients that received nivolumab-chemotherapy than the 19 patients received chemotherapy alone (56% versus 35%). Those patients with ctDNA clearance had longer EFS (HR 0.6) and higher pCR rate (HR 0.13) compared to those without ctDNA clearance. A recent prospective study enrolled 261 patients with stages I to III NSCLC who underwent surgical resection and evaluated the prognostic value of ctDNA-MRD detection during the surveillance of NSCLC. Those patients who maintained longitudinal undetectable MRD for more than 18 months could be classified as the potentially cured population. However, most current ctDNA analysis methods lack adequate sensitivity for residual disease detection during and after completion of treatment in cancer patients, along with high cost [117]. Patients with early-stage NSCLC had lower plasma ctDNA compared to those with metastatic NSCLC for tumor genotyping. Furthermore, some patients with ctDNA clearance still had cancer recurrence. Thus, highly sensitive blood tests are needed to detect low quantity of systemic micrometastasis in the blood after surgery. Extracellular vesicles such as exosomes play a role in cancer progression by transferring bioactive molecules to non-tumorigenic and tumorigenic recipient cells [118][119][120]. Tumor-derived exosomes isolated from the plasma of patients contain high quality nucleic acids (DNA, RNA, and miRNA), which is suitable for tumor genomic typing [121]. Thus, detecting tumor-derived exosomes in patients' plasma after surgery might serve as a surrogate for residual micrometastasis. Furthermore, integrins mediate the organ-specific metastases of circulating tumor cells and exosomes [118,122,123]. Ongoing studies are evaluating if ctDNA detection may inform a personalized approach to duration of PD-(L)1 treatment in patients with longterm response. For those patients with undetectable ctDNA, they may be able to safely discontinue treatment. Additional work will be needed to determine the optimal timepoint (e.g., 12-24 months after initiating treatment) at which to query ctDNA to guide such a decision.
Additionally, lymphocyte counts and immunophenotyping of T-cell and B-cell have been used to assess the immune status and are prognostic biomarkers. Low levels of lymphocytes in the blood could indicate an increased risk for death [124]. In NSCLC patients who had MPR, lower baseline levels of T cells and NK cells, but after neoadjuvant therapy, these patients experienced an expansion of NK cells and granulocytes and increased abundance of DC and B cells in lymph nodes, as well as decreased abundance of monocytes, suggesting that immunotherapy plays a key role in preoperative activation of tumor-specific immune killing [125]. Several studies have shown that pre-treatment low ALCs (< 600-1200 cells/µL) were associated with decreased PFS and OS to ICI in NSCLC patients [126][127][128][129]. Dynamic changes of ALCs after ICI treatment were also associated with clinical response. Post-ICI treatment low ALCs (< 700-900 cells/µL) were associated with decreased PFS and OS in patients with advanced solid cancer types including NSCLC [127,130]. Immunophenotypic analysis of circulating immune cells revealed increases in circulating proliferating CD4 + and CD8 + T cells at 2 weeks after durvalumab treatment [131]. Assessing the function of peripheral T-cell subclones, particularly the T-cell receptor (TCR) clonality and activity to clonal neoantigens, have also been explored as a predictive biomarker for response to ICI [131,132]. Furthermore, high derived neutrophilto-lymphocyte ratio (dNLR) was associated with poor prognosis in patients with advanced NSCLC treated with durvalumab [133]. Figure 7 summarizes the emerging biomarkers of tumor tissue, blood, host, and radiology for neoadjuvant therapy in NSCLC. Further studies are needed to evaluate the role of these blood biomarkers in guiding neoadjuvant/adjuvant therapy.

Summary and perspectives
Neoadjuvant therapies hold great promise to improve surgical and pathological outcomes, reduce local and systemic recurrences, guide postoperative therapy, and improve cure rates in patients with resectable NSCLC. The biomarker-driven neoadjuvant treatment extends the precision oncology from advanced stage  to early-stage NSCLC. This is particularly important for those stage IIIA patients with N2 disease who are mostly (61%) treated with chemotherapy and/or radiation [10]. Table 7 summarizes the advantages and disadvantages of neoadjuvant chemoimmunotherapy in patients with resectable NSCLC. The preoperative evaluation and timely biomarker testing, in the era of precision neoadjuvant therapy for early-stage NSCLC, requires a paradigm shift. This paradigm shift will require the collaboration of thoracic surgeons, medical oncologists, interventional pulmonologists, radiologists, and pathologists. In addition, the emerging neoadjuvant chemoimmunotherapy has brought several new radiographic, pathological, and clinical endpoints that would need further evaluation. Further research studies are required to compare neoadjuvant and adjuvant treatment, optimize biomarker guided treatment selection, and define the treatment duration to improve the survival of early-stage NSCLC.